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Characterization of a novel marine unicellular alga, Pseudoneochloris sp. strain NKY372003 as a high carbohydrate producer
Journal of Bioscience and Bioengineering VOL. xxx No. xxx, xxx, xxxx www.elsevier.com/locate/jbiosc
Characterization of a novel marine unicellular alga, Pseudoneochloris sp. strain NKY372003 as a high carbohydrate producer Tsuyoshi Aketo,1, 2 Rina Hashizume,1 Yusuke Yabu,1 Yumiko Hoshikawa,1 Daisuke Nojima,1 Yoshiaki Maeda,1 Tomoko Yoshino,1 Hiroyuki Takano,2 and Tsuyoshi Tanaka1, * Division of Biotechnology and Life Science, Institute of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan1 and Central Research Laboratory, Taiheiyo Cement Corporation, 2-4-2 Osaku, Sakura City, Chiba 285-8655, Japan2 Received 2 October 2019; accepted 21 December 2019 Available online xxx
Production of biofuels and fine chemicals from biomass-derived carbohydrates through biorefinery attracts much attention because it is recognized as an environmentally friendly process. Microalgae can serve as promising carbohydrate producers for biorefinery rather than woody and crop biomass due to high biomass productivity, high CO2 fixation, and no competition with food production. However, microalgae with high carbohydrate productivity have not been well investigated despite intensive studies of microalgal lipid production. In this study, the carbohydrate production of Pseudoneochloris sp. strain NKY372003 isolated as a high carbohydrate producer, was investigated. Cultivation conditions with various combinations of nutrient contents and photon flux density were examined to maximize the biomass and carbohydrate productivities. At the optimal condition, the biomass and carbohydrate production of this strain reached 8.11 ± 0.37 g/L and 5.5 ± 0.2 g/L, respectively. As far as we know, this is the highest carbohydrate production by microalgae among ever reported. Cell staining with Lugol’s solution visualized intracellular starch granules. Because algal starch can be converted to biofuels and building blocks of fine chemicals, Pseudoneochloris sp. NKY372003 will be a promising candidate for production of fermentable carbohydrates towards biofuels and fine chemicals production. Ó 2020, The Society for Biotechnology, Japan. All rights reserved. [Key words: Marine unicellular algae; Carbohydrate producer; Pseudoneochloris sp.; Fine chemicals; Biorefinery]
Various important fuels and materials, such as raw materials of plastics, are currently produced through oil refinery process. However, owing to the depletion of petroleum, alternative and renewable carbon sources became highly desired. Woody and crop biomass have been recognized as a first generation alternative carbon sources which can be converted to valuable compounds through biorefinery technology. Advantages of biorefinery process over conventional oil refinery are low environmental load and decreasing dependency on fossil fuels. In particular, photosynthetic property of plants can contribute to decrease in CO2 emission. Nonetheless, cellulose and hemicellulose which are abundantly contained in plant biomass are less useful in biorefinery because of their robust and persistent molecular structures. Subsequently, algal biomass begun to attract more attentions as next generation alternatives in biorefinery. Some algal species accumulate high amount of carbohydrates (e.g., starch and glycogen as energystorage compounds, and sulfated polysaccharide as a component of algal cell walls), and less cellulose and hemicellulose unlike plant biomass. For example, macroalgae Ulvales (Chlorophyta) such as Ulva lactuca produce high amount of sulfated polysaccharide called ulvan (1), which is currently recognized as a promising substance in biorefinery applications (2,3).
* Corresponding author. Tel.: þ81 42 388 7401; fax: þ81 42 385 7713. E-mail address: [email protected] (T. Tanaka).
By contrast, unicellular microalgae with high carbohydrate producing property have not been comprehensively screened in spite of intensive studies of microalgal lipid production. Microalgae have a number of additional advantages as promising hosts of carbohydrates, for example high CO2 fixation properties, high growth rates and no competition with food and feed productions (4,5). Up to date, production of biofuels (e.g., ethanol and methane) has been studied using some microalgae (6) such as Chlorella vulgaris (7,8) and Chlamydomonas reinhardtii (9,10). Recently, novel biorefinery research using microalgal biomass has just launched to produce the specific compounds, such as methyl levulinate, methyl lactate (11) and succinate (12), which can be initial building blocks of a wide range of fine chemicals (e.g., pharmaceuticals and bioplastics). According to these circumstance, exploration of novel microalgae which produce high amount of carbohydrates will contribute to development of more efficient and affordable biorefinery processes. In our laboratory, we have screened microalgal strains producing high amount of carbohydrate compounds, and isolated a promising microalga strain NKY372003. In this study, we performed morphological observation and genotyping analysis, which indicates that this strain belongs to the algal genus Pseudoneochloris. Subsequently, we optimized the culture condition of this strain for high carbohydrate production by changing nutrient compositions of culture medium (e.g., nitrate and phosphate) and photon flux density. Based on the obtained results, we discussed
1389-1723/$ e see front matter Ó 2020, The Society for Biotechnology, Japan. All rights reserved. https://doi.org/10.1016/j.jbiosc.2019.12.010
Please cite this article as: Aketo, T et al., Characterization of a novel marine unicellular alga, Pseudoneochloris sp. strain NKY372003 as a high carbohydrate producer, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.12.010
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the potential of this strain as a host of carbohydrate production for biorefinery application.
MATERIALS AND METHODS Used strain Microalgal strains were isolated from seawater obtained from sites on the coast of Yakushima island, which is one of the Osumi islands in Kagoshima prefecture, Japan. Among 292 strains isolated, strain NKY372003 was selected as the highest carbohydrate producer for further study here. This strain was maintained in IMK medium (Nihon Pharmaceutical, Osaka, Japan) dissolved in artificial seawater in Erlenmeyer flasks (50 ml) at 100 mmol photons/m2/s and 30 C under the shaking condition. Microscopic analysis The morphologies of the cultured cells were analyzed by a differential interference contrast microscope (BX51; Olympus, Tokyo, Japan). Twenty cells were randomly selected and the diameters were measured by an image analysis software, ImageJ (13). To visualize intracellular starch granules, Lugol’s solution was prepared. Iodine crystals (50 mg) were dissolved in 1 ml of 100 mg/ ml potassium iodide solution. Subsequently, 10 ml of Lugol’s solution was added to 1 ml of cell suspension. Then, the cell suspension was heated at 98 C for 5 min to complete the staining (14). Phylogenetic analysis Strain NKY372003 was identified after analysis of the 18S rDNA sequence. Nucleic acid extraction was performed with DNeasy Tissue Kit (Qiagen, Venlo, Netherlands) in accordance with the manufacturer’s instructions. The 18S rDNA was amplified by PCR using a pair of universal primers, ss5 (50 -GGTGA TCCTG CCAGT AGTCA TATGC TTG-30 ) and ss3 (50 -GATCC TTCCG CAGGT TCACC TACGG AAACC-30 ) (15) and Takara LA Taq DNA polymerase (Takara Bio Inc., Shiga, Japan). The amplified fragments were inserted into pCR4-TOPO vector (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA). The resulting plasmids were sequenced using the following primers: Forward primers, T3 promoter-1 (50 ATTAA CCCTC ACTAA AGGGA-30 ), T7 promoter-1 (50 -TAATA CGACT CACTA TAGGG30 ) and 18SU467F (50 -ATCCA AGGAA GGCAG CAGGC-30 ); Reverse primers, 18SU1310R (50 -CTCCA CCAAC TAAGA ACGGC-30 ) (16). Sequence of 18S rDNA was determined by Fasmac DNA Sequence Service (Kanagawa, Japan). Sequence homologies were analyzed with BLAST. Evaluation of cell growth For the growth experiment, the full strength IMK nutrients, containing 2.4 mM nitrate and 0.038 mM phosphate, were dissolved in the artificial seawater (Osaka Yakken Co., Ltd., Osaka, Japan). We also prepared the IMK media with higher concentration of nitrate (4.7, 11.8 or 23.5 mM) by adding NaNO3, or higher concentration of phosphate (0.38 or 0.76 mM) by adding K2HPO4 and Na2HPO4 (weight ratio, 7:25, same with IMK medium). In addition, the condensed IMK media, 2IMK, 10IMK and 20IMK media, in which all nutrients were concentrated by 2-, 10-, and 20-fold as compare to the standard IMK medium, were prepared. Strain NKY372003 at the initial concentration of 1.0 105 cells/ml was cultured in each condition for 10 days in flat-shaped flasks (500 ml) at 30 C. Concentrated CO2 (2% in air) was supplied at the rate of 0.8 l/l/min for photoautotrophic culture. Furthermore, the effect of photon flux density on growth was evaluated. The cultured cells were collected at every 24 h, and growth was monitored by cell counting using hemocytometer. Three biological repetitions were analyzed for each sample. Measurement of biomass production, and carbohydrate and protein contents The cultured cells (5 ml) were collected at every 24 h. After centrifugation at 9000 g, 4 C for 10 min, the pellet was washed with 10 ml of deionized water. The algal biomass was lyophilized to measure the dry weight. Phenol-sulfuric acid method was employed to measure saccharide content (17). After the lyophilized cells were suspended in 500 ml of ultrapure water at the concentration of 0.5 mg/ml, 500 ml of 5% (w/v) phenol solution and 2.5 ml of concentrated sulfuric acid were added to the suspension. The samples were incubated for 30 min at room temperature. Subsequently, the absorbance at 490 nm was measured using a microplate reader (SH-9000; Corona Electric, Ibaraki, Japan). Protein quantity was analyzed by BCA Protein assay kit (Thermo Fisher Scientific). Absorbance at 562 nm was measured using the microplate reader. Measurement of nitrate concentration by HPLC Culture media were filtered using a membrane filter (Millex Filter Unit 0.22 mm). Nitrate concentrations in the filtered culture medium were determined using a Shimadzu Prominence HPLC system (Shimadzu Scientific Instruments, Kyoto, Japan) equipped with an anion exchange column, TSKgel IC column (Tosoh Co., Tokyo, Japan). TSKgel eluent ICAnion-A was used as a mobile phase at the flow rate of 1.2 ml/min to elute nitrate ion, and absorbance at 220 nm was measured by a UV detector (Shimadzu Scientific Instruments, Kyoto, Japan).
RESULTS AND DISCUSSION Characterization of strain NKY372003 by genotypic and morphological analyses As a result of the screening from 292 isolates, strain NKY372003 showed fast growth and the highest
carbohydrate content. Then, we examined the genotypic and morphological characteristics of this strain. Strain NKY372003 was identified based on 18S rDNA sequences (DDBJ/EMBL/GenBank accession number: LC505539), and displayed 96.95% of sequence identity with the 18S rDNA sequence of the unicellular green alga, Pseudoneochloris marina. Microscopic analysis indicated that vegetative cells of strain NKY372003 were spherical and nonmotile. Cell size was drastically changed during the growth phase, ranging from 3 mm to 15 mm in diameter (Figs. 1 and S1). Zoospores were produced in mature vegetative cells (Fig. S2A), and two flagella were located at the apex of the zoospore (Fig. S2B). Furthermore, Lugol staining indicates that starch granules were located and accumulated in the chloroplast (Fig. S3). These results were in agreement with the morphological data of P. marina (18). Therefore, this strain was tentatively identified as Pseudoneochloris sp. in this study.
Growth characteristics of Pseudoneochloris sp. NKY372003 at different nitrate and phosphate concentrations Fig. 2 shows the growth curves of Pseudoneochloris sp. NKY372003 in IMK medium (2.4 mM nitrate) at different photon flux densities. The final cell concentrations increased with increasing photon flux densities (Fig. 2A), and nitrates in the supernatants were completely consumed within 7 days in all the conditions (Fig. 2B). Although the final cell densities were different, the final biomass yield did not change markedly (Table S1). Especially, the biomass production at 800 mmol photons/m2/s was lower than those at 100 and 300 photons/m2/s, although the cell concentration at 800 mmol photons/m2/s was significantly higher than those at lower photon flux densities. This inconsistency could be due to smaller cell size at 800 mmol photons/m2/s than those at lower photon flux densities as mentioned above (Fig. S1). Decrease in cell size at 800 mmol photons/m2/s might be because of the phototoxicity at higher light intensities. These results indicated that light intensity was not a limiting factor for growth of strain NKY372003 in the standard IMK medium. Therefore, the effects of nutrients on algal growth was evaluated in the following experiments. Because the nitrogen and phosphorous (2.4 mM nitrate and 0.038 mM phosphate are contained in IMK medium) are essential elements for the growth of microalgae, effects of these nutrients on growth were firstly examined by adding them to standard IMK medium. It should be noted that pH values of the culture did not change largely. The initial pH of IMK medium was 7.27. After Pseudoneochloris sp. NKY372003 was cultivated in the IMK medium at 100 mmol photons/m2/s for 10 days, the pH value of the culture was 7.41 0.10. This could be due to the buffer action of the seawater-based culture medium. The growth of Pseudoneochloris sp. NKY372003 under the increased nitrate concentration (4.7 mM) at different light intensities (500 or 800 mmol photons/m2/s) was monitored. An increasing trend in cell concentrations was observed as increased nitrate concentration and light intensity (Fig. 3A). However, complete uptake of nitrate was not observed even at 4.7 mM, and the reductions of nitrate concentrations during the cultivation were comparable levels in all the tested conditions (Fig. 3B). As mentioned above, the cell concentrations were not exactly correlated to the biomass production in this experiment. Correspondingly, the biomass production did not change markedly by increasing the nitrate concentration and photon flux density (Table S2). Furthermore, the similar tendencies (i.e., increase in cell concentrations, but slight change in biomass production) were observed when increased phosphate concentrations were employed for the culture of Pseudoneochloris sp. NKY372003 (Table S3). Because sufficient CO2 (2%) was supplied in all the tested
Please cite this article as: Aketo, T et al., Characterization of a novel marine unicellular alga, Pseudoneochloris sp. strain NKY372003 as a high carbohydrate producer, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.12.010
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FIG. 2. Growth curve (A) and nitrate consumption (B) of Pseudoneochloris sp. NKY372003 cultured in standard IMK medium (2.4 mM nitrate and 0.038 mM phosphate) under different photon flux density (100e800 mmol photons/m2/s). Error bars indicate standard deviation (n ¼ 3).
FIG. 1. Microscope images of vegetative cells of Pseudoneochloris sp. NKY372003 cultivated in IMK medium for 10 days. Scale bars are 10 mm.
conditions, the medium components other than nitrogen and phosphorus were the limiting factor(s) of the biomass production. Evaluation of biomass production of Pseudoneochloris sp. NKY372003 in enriched culture media Sole increase in nitrate or phosphate in IMK medium did not significantly improve the biomass production of Pseudoneochloris sp. NKY372003. Then, in order to enhance the biomass production, enriched culture
media (i.e., concentrated 2 and 10IMK media) were tested under various light intensities. Only a representative experiment is shown in Fig. 4. Fig. 4A shows the growth curves of Pseudoneochloris sp. NKY372003 in 10IMK medium at 500 or 800 mmol photons/ m2/s of photon flux density. The cell concentrations in 10IMK medium was approximately twenty times higher than those in the standard IMK medium (Fig. 3A). It should be noted that the Yaxis in Fig. 4A was tenfold increase compared with that of Fig. 3A. Although much higher concentration of nitrate (23.5 mM) was contained in the condensed 10IMK medium, almost complete consumption of nitrate in the supernatants was observed in 5 days at both light conditions (Fig. 4B). As mentioned above,
Please cite this article as: Aketo, T et al., Characterization of a novel marine unicellular alga, Pseudoneochloris sp. strain NKY372003 as a high carbohydrate producer, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.12.010
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FIG. 3. Growth curve (A) and nitrate consumption (B) of Pseudoneochloris sp. NKY372003 cultured under different nitrate concentrations (2.4 or 4.7 mM) and photon flux density (500 or 800 mmol photons/m2/s). Error bars indicate standard deviation (n ¼ 3).
4.7 mM nitrate ions were not completely consumed in the standard IMK medium (Fig. 3B). By contrast, in 10IMK medium, additional nutrients could significantly accelerate the growth of Pseudoneochloris sp. NKY372003, and correspondingly enhance the uptake of nitrate ions in the enriched culture medium. The key components in the medium for this phenomenon should be elucidated in the future. The biomass production data using various media under different photon flux densities is summarized in Table 1. The use of 2IMK almost double in the biomass production compared with the standard IMK medium at 500 and 800 mmol photons/m2/s. Similarly, the use of 10IMK resulted in approximately 10- and 12-
J. BIOSCI. BIOENG.,
FIG. 4. Growth curve (A) and nitrate consumption (B) of Pseudoneochloris sp. NKY372003 in 10IMK medium at 500 or 800 mmol photons/m2/s. Error bars indicate standard deviation (n ¼ 3).
fold higher biomass production at 500 and 800 mmol photons/m2/s, respectively. These results indicate that the biomass production of Pseudoneochloris sp. NKY372003 was nutrient-dependent at these TABLE 1. Biomass production of Pseudoneochloris sp. NKY372003 cultured in the enriched IMK media at different photon flux density. Medium
Photon flux density (mmol photons/m2/s)
IMK 2IMK 10IMK IMK 2IMK 10IMK 10IMK
500 500 500 800 800 800 1300
Biomass production (g/L) 0.65 1.53 6.65 0.65 1.56 8.11 6.20
0.15 0.09 0.49 0.15 0.02 0.37
Please cite this article as: Aketo, T et al., Characterization of a novel marine unicellular alga, Pseudoneochloris sp. strain NKY372003 as a high carbohydrate producer, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.12.010
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obviously repressed the cell growth in 10IMK, resulting in lower biomass production (6.20 g/L). This might be also caused by the phototoxicity at higher light intensities. In addition, 20IMK was used for the growth test, however, no cell growth was observed (data not shown). In microalgae, cell growth can be inhibited due to excessive nutrients such as nitrate, nitrite, and ammonium (19,20). In C. vulgaris, growth inhibition was observed at more than 29 mM nitrate (20). The increase of intracellular nitrate may induce high accumulation of nitrite and/or ammonium by nitrate reductase, resulting in the growth inhibition by the accumulated toxic compounds (20). The similar growth inhibition could occur in this experiment because the condensed 20IMK medium contained much higher nitrate concentration (47 mM nitrate) than that previously reported. Based on the above results, carbohydrate production in strain NKY372003 was further investigated in 10IMK medium in the following experiments.
FIG. 5. Time course variations of carbohydrate (closed bars) and protein (open bars) contents, and biomass production (closed circles) of Pseudoneochloris sp. NKY372003 in 10IMK medium at (A) 500 or (B) 800 mmol photons/m2/s. Error bars indicate standard deviation (n ¼ 3).
conditions, although the nutrients which indeed limited the growth were not specified in this study. The maximum biomass production was 8.11 0.37 g/L at 800 mmol photons/m2/s when 10IMK was used. Higher light (1300 mmol photons/m2/s)
Evaluation of carbohydrate production of Pseudoneochloris sp. NKY372003 Fig. 5 shows time-courses variations of carbohydrate and protein contents, and biomass productions in 10IMK medium at 500 or 800 mmol photons/m2/s. Remarkable carbohydrate accumulation was observed after day 6. Because nitrate consumption in 10IMK medium was almost completely consumed at day 5 (Fig. 4B), the carbohydrate accumulation could be induced by nitrogen-deficient stress. It was frequently reported that nitrogen starvation induced carbohydrate accumulation in microalgal cells (21,22). In contrast, the protein contents tended to decrease with time. Nitrogen is an important component for the synthesis of amino acids (proteins), therefore, the tradeeoff relationship between carbohydrate and protein contents could be a response to the nitrogen deficiency, generally observed in microalgae (23,24). It should be noted that obvious decrease in biomass production was repeatedly observed at day 7 at 800 mmol photons/m2/s (Fig. 5B). The phenomenon will be elucidated in detail in the future, but the zoospore formation (Fig. S2A) from vegetative cells will be a possible reason for the weight loss. Both carbohydrate content and biomass production were maximum at 800 mmol photons/m2/s (67.8 7.0%, and 8.11 0.37 g/L, respectively) at day 10. The resulting carbohydrate production reached to 5.5 0.2 g/L. Recently, biochemical compositions of P. marina, the closely related microalga, cultivated under various conditions was reported (21). According to this literature, maximal biomass production and carbohydrate content of P. marina reached 2.02 0.21 g/L and 53.8% in 7 days, respectively, suggesting that carbohydrate production in P. marina was less than 1.09 g/L. Carbohydrate production of representative microalgae is summarized in Table 2. As far as we know, carbohydrate production by Pseudoneochloris sp. NKY372003 was the highest level among ever reported. The carbohydrate productivity per unit time of Pseudoneochloris sp. NKY372003 was 0.55 0.08 g/L/day, which were higher than that of P. marina (<0.16 g/L/day) (21), and comparable to that of Tetraselmis subcordiformis (0.20e0.62 g/L/day) (22).
TABLE 2. Comparison of biomass and carbohydrate production among various microalgal species. Biomass production (g/L) Chlorella vulgaris Chlamydomonas reinhardtii Scenedesmus obliquus Arthrospira platensis (Spirulina platensis) Synechococcus sp. PCC7002 Tetraselmis subcordiformis Pseudoneochloris marina Pseudoneochloris sp. NKY372003
Carbohydrate production (g/L)
Reference
0.5e7.3 1.5e2.4 2.6e4.0 2.0e2.2
0.3e3.6 0.8e1.2 1.3e1.9 1.2e1.3
23,25,26 27,28 23,29 30
3.0 4.5e6.0 1.33e2.02 8.11 0.37
1.8 1.8e3.0 <1.09 5.5 0.2
31 22,32 21 This study
Please cite this article as: Aketo, T et al., Characterization of a novel marine unicellular alga, Pseudoneochloris sp. strain NKY372003 as a high carbohydrate producer, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.12.010
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As mentioned above, Pseudoneochloris sp. NKY37200 accumulated starch granules intracellularly. Starch can be easily converted to fermentable monosaccharides like glucose, and subsequently such monosaccharides can be transformed into bioethanol and biobutanol by a fermentation process. Furthermore, recently, algal starch can be successfully fermented to building blocks, such as succinate, methyl levulinate or methyl lactate, useful for the synthesis of fine chemicals (11,12). Therefore, Pseudoneochloris sp. NKY372003 will be a promising candidate for production of fermentable sugar towards biofuels and fine chemicals production. In conclusion, microalga Pseudoneochloris sp. NKY372003 was newly identified as a high carbohydrate producer. Elevation of entire concentration of IMK medium drastically increased the biomass production, which reached the maximal value of 8.11 0.37 g/L when 10IMK medium were used for cultivation at 800 mmol photons/m2/s. In this condition, carbohydrate content and production were 67.8 7.0% and 5.5 0.2 g/L, respectively. To the best of our knowledge, this is the highest carbohydrate production by microalgae. These results indicate that Pseudoneochloris sp. NKY372003 is a promising host of carbohydrate production for biorefinery applications. Supplementary data to this article can be found online at https://doi.org/10.1016/j.jbiosc.2019.12.010. ACKNOWLEDGMENTS This work was partially supported by JSPS KAKENHI Grant-inAid for Scientific Research B [grant number 17H03465]. References 1. Lahaye, M. and Robic, A.: Structure and functional properties of ulvan, a polysaccharide from green seaweeds, Biomacromolecules, 8, 1765e1774 (2007). 2. Bikker, P., van Krimpen, M. M., van Wikselaar, P., Houweling-Tan, B., Scaccia, N., van Hal, J. W., Huijgen, W. J., Cone, J. W., and LopezContreras, A. M.: Biorefinery of the green seaweed Ulva lactuca to produce animal feed, chemicals and biofuels, J. Appl. Phycol., 28, 3511e3525 (2016). 3. Glasson, C. R., Sims, I. M., Carnachan, S. M., de Nys, R., and Magnusson, M.: A cascading biorefinery process targeting sulfated polysaccharides (ulvan) from Ulva ohnoi, Algal Res., 27, 383e391 (2017). 4. Chisti, Y.: Biodiesel from microalgae, Biotechnol. Adv., 25, 294e306 (2007). 5. Maeda, Y., Yoshino, T., Matsunaga, T., Matsumoto, M., and Tanaka, T.: Marine microalgae for production of biofuels and chemicals, Curr. Opin. Biotechnol., 50, 111e120 (2018). 6. Chen, C. Y., Zhao, X. Q., Yen, H. W., Ho, S. H., Cheng, C. L., Lee, D. J., Bai, F. W., and Chang, J. S.: Microalgae-based carbohydrates for biofuel production, Biochem. Eng. J., 78, 1e10 (2013). 7. Kim, K. H., Choi, I. S., Kim, H. M., Wi, S. G., and Bae, H. J.: Bioethanol production from the nutrient stress-induced microalga Chlorella vulgaris by enzymatic hydrolysis and immobilized yeast fermentation, Bioresour. Technol., 153, 47e54 (2014). 8. Moncada, J., Jaramillo, J. J., Higuita, J. C., Younes, C., and Cardona, C. A.: Production of bioethanol using Chlorella vulgaris cake: a technoeconomic and environmental assessment in the colombian context, Ind. Eng. Chem. Res., 52, 16786e16794 (2013). 9. Choi, S. P., Nguyen, M. T., and Sim, S. J.: Enzymatic pretreatment of Chlamydomonas reinhardtii biomass for ethanol production, Bioresour. Technol., 101, 5330e5336 (2010). 10. Osanai, T., Shirai, T., Iijima, H., Nakaya, Y., Okamoto, M., Kondo, A., and Hirai, M. Y.: Genetic manipulation of a metabolic enzyme and a transcriptional regulator increasing succinate excretion from unicellular cyanobacterium, Front. Microbiol., 6, 1064 (2015). 11. Yamaguchi, S., Kawada, Y., Yuge, H., Tanaka, K., and Imamura, S.: Development of new carbon resources: production of important chemicals from algal residue, Sci. Rep., 7, 855 (2017).
12. Lee, J., Sim, S. J., Bott, M., Um, Y., Oh, M. K., and Woo, H. M.: Succinate production from CO2-grown microalgal biomass as carbon source using engineered Corynebacterium glutamicum through consolidated bioprocessing, Sci. Rep., 4, 5819 (2014). 13. Schneider, C. A., Rasband, W. S., and Eliceiri, K. W.: NIH Image to ImageJ: 25 years of image analysis, Nat. Methods, 9, 671e675 (2012). 14. Takeshita, T., Takeda, K., Ota, S., Yamazaki, T., and Kawano, S.: A simple method for measuring the starch and lipid contents in the cell of microalgae, Cytologia, 80, 475e481 (2015). 15. Rowan, R. and Powers, D. A.: Molecular genetic identification of symbiotic dinoflagellates (zooxanthellae), Marine ecology progress series, Oldendorf, 71, 65e73 (1991). 16. Suzuki, N., Murakami, K., Takeyama, H., and Chow, S.: Molecular attempt to identify prey organisms of lobster phyllosoma larvae, Fish. Sci., 72, 342e349 (2006). 17. DuBois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F.: Colorimetric method for determination of sugars and related substances, Anal. Chem., 28, 350e356 (1956). 18. Watanabe, S., Himizu, A., Lewis, L. A., Floyd, G. L., and Fuerst, P. A.: Pseudoneochloris marina (Chlorophyta), a new coccoid ulvophycean alga, and its phylogenetic position inferred from morphological and molecular data, J. Phycol., 36, 596e604 (2000). 19. Markou, G., Vandamme, D., and Muylaert, K.: Microalgal and cyanobacterial cultivation: the supply of nutrients, Water Res., 65, 186e202 (2014). 20. Jeanfils, J., Canisius, M., and Burlion, N.: Effect of high nitrate concentrations on growth and nitrate uptake by free-living and immobilized Chlorella vulgaris cells, J. Appl. Phycol., 5, 369e374 (1993). 21. Gonçalves, C. F., Menegol, T., and Rech, R.: Biochemical composition of green microalgae Pseudoneochloris marina grown under different temperature and light conditions, Biocatal. Agric. Biotechnol., 18, 101032 (2019). 22. Yao, C., Ai, J., Cao, X., Xue, S., and Zhang, W.: Enhancing starch production of a marine green microalga Tetraselmis subcordiformis through nutrient limitation, Bioresour. Technol., 118, 438e444 (2012). 23. Ho, S. H., Chen, C. Y., and Chang, J. S.: Effect of light intensity and nitrogen starvation on CO2 fixation and lipid/carbohydrate production of an indigenous microalga Scenedesmus obliquus CNW-N, Bioresour. Technol., 113, 244e252 (2012). 24. Siaut, M., Cuine, S., Cagnon, C., Fessler, B., Nguyen, M., Carrier, P., Beyly, A., Beisson, F., Triantaphylides, C., Li-Beisson, Y., and Peltier, G.: Oil accumulation in the model green alga Chlamydomonas reinhardtii: characterization, variability between common laboratory strains and relationship with starch reserves, BMC Biotechnol., 11, 7 (2011). 25. Illman, A. M., Scragg, A. H., and Shales, S. W.: Increase in Chlorella strains calorific values when grown in low nitrogen medium, Enzym. Microb. Technol., 27, 631e635 (2000). 26. Liang, Y., Sarkany, N., and Cui, Y.: Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions, Biotechnol. Lett., 31, 1043e1049 (2009). 27. Kim, M. S., Baek, J. S., Yun, Y. S., Sim, S. J., Park, S., and Kim, S. C.: Hydrogen production from Chlamydomonas reinhardtii biomass using a two-step conversion process: anaerobic conversion and photosynthetic fermentation, Int. J. Hydrogen Energy, 31, 812e816 (2006). 28. Nguyen, M. T., Choi, S. P., Lee, J., Lee, J. H., and Sim, S. J.: Hydrothermal acid pretreatment of Chlamydomonas reinhardtii biomass for ethanol production, J. Microbiol. Biotechnol., 19, 161e166 (2009). 29. Ho, S. H., Chen, C. Y., Yeh, K. L., Chen, W. M., Lin, C. Y., and Chang, J. S.: Characterization of photosynthetic carbon dioxide fixation ability of indigenous Scenedesmus obliquus isolates, Biochem. Eng. J., 53, 57e62 (2010). 30. Markou, G., Angelidaki, I., Nerantzis, E., and Georgakakis, D.: Bioethanol production by carbohydrate-enriched biomass of Arthrospira (Spirulina) platensis, Energies, 6, 3937e3950 (2013). 31. Mollers, K. B., Cannella, D., Jorgensen, H., and Frigaard, N. U.: Cyanobacterial biomass as carbohydrate and nutrient feedstock for bioethanol production by yeast fermentation, Biotechnol. Biofuels, 7, 64 (2014). 32. Yao, C. H., Ai, J. N., Cao, X. P., and Xue, S.: Characterization of cell growth and starch production in the marine green microalga Tetraselmis subcordiformis under extracellular phosphorus-deprived and sequentially phosphorus-replete conditions, Appl. Microbiol. Biotechnol., 97, 6099e6110 (2013).
Please cite this article as: Aketo, T et al., Characterization of a novel marine unicellular alga, Pseudoneochloris sp. strain NKY372003 as a high carbohydrate producer, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.12.010
Characterization of a novel marine unicellular alga, Pseudoneochloris sp. strain NKY372003 as a high carbohydrate producer Tsuyoshi Aketo,1, 2 Rina Hashizume,1 Yusuke Yabu,1 Yumiko Hoshikawa,1 Daisuke Nojima,1 Yoshiaki Maeda,1 Tomoko Yoshino,1 Hiroyuki Takano,2 and Tsuyoshi Tanaka1, * Division of Biotechnology and Life Science, Institute of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan1 and Central Research Laboratory, Taiheiyo Cement Corporation, 2-4-2 Osaku, Sakura City, Chiba 285-8655, Japan2 Received 2 October 2019; accepted 21 December 2019 Available online xxx
Production of biofuels and fine chemicals from biomass-derived carbohydrates through biorefinery attracts much attention because it is recognized as an environmentally friendly process. Microalgae can serve as promising carbohydrate producers for biorefinery rather than woody and crop biomass due to high biomass productivity, high CO2 fixation, and no competition with food production. However, microalgae with high carbohydrate productivity have not been well investigated despite intensive studies of microalgal lipid production. In this study, the carbohydrate production of Pseudoneochloris sp. strain NKY372003 isolated as a high carbohydrate producer, was investigated. Cultivation conditions with various combinations of nutrient contents and photon flux density were examined to maximize the biomass and carbohydrate productivities. At the optimal condition, the biomass and carbohydrate production of this strain reached 8.11 ± 0.37 g/L and 5.5 ± 0.2 g/L, respectively. As far as we know, this is the highest carbohydrate production by microalgae among ever reported. Cell staining with Lugol’s solution visualized intracellular starch granules. Because algal starch can be converted to biofuels and building blocks of fine chemicals, Pseudoneochloris sp. NKY372003 will be a promising candidate for production of fermentable carbohydrates towards biofuels and fine chemicals production. Ó 2020, The Society for Biotechnology, Japan. All rights reserved. [Key words: Marine unicellular algae; Carbohydrate producer; Pseudoneochloris sp.; Fine chemicals; Biorefinery]
Various important fuels and materials, such as raw materials of plastics, are currently produced through oil refinery process. However, owing to the depletion of petroleum, alternative and renewable carbon sources became highly desired. Woody and crop biomass have been recognized as a first generation alternative carbon sources which can be converted to valuable compounds through biorefinery technology. Advantages of biorefinery process over conventional oil refinery are low environmental load and decreasing dependency on fossil fuels. In particular, photosynthetic property of plants can contribute to decrease in CO2 emission. Nonetheless, cellulose and hemicellulose which are abundantly contained in plant biomass are less useful in biorefinery because of their robust and persistent molecular structures. Subsequently, algal biomass begun to attract more attentions as next generation alternatives in biorefinery. Some algal species accumulate high amount of carbohydrates (e.g., starch and glycogen as energystorage compounds, and sulfated polysaccharide as a component of algal cell walls), and less cellulose and hemicellulose unlike plant biomass. For example, macroalgae Ulvales (Chlorophyta) such as Ulva lactuca produce high amount of sulfated polysaccharide called ulvan (1), which is currently recognized as a promising substance in biorefinery applications (2,3).
* Corresponding author. Tel.: þ81 42 388 7401; fax: þ81 42 385 7713. E-mail address: [email protected] (T. Tanaka).
By contrast, unicellular microalgae with high carbohydrate producing property have not been comprehensively screened in spite of intensive studies of microalgal lipid production. Microalgae have a number of additional advantages as promising hosts of carbohydrates, for example high CO2 fixation properties, high growth rates and no competition with food and feed productions (4,5). Up to date, production of biofuels (e.g., ethanol and methane) has been studied using some microalgae (6) such as Chlorella vulgaris (7,8) and Chlamydomonas reinhardtii (9,10). Recently, novel biorefinery research using microalgal biomass has just launched to produce the specific compounds, such as methyl levulinate, methyl lactate (11) and succinate (12), which can be initial building blocks of a wide range of fine chemicals (e.g., pharmaceuticals and bioplastics). According to these circumstance, exploration of novel microalgae which produce high amount of carbohydrates will contribute to development of more efficient and affordable biorefinery processes. In our laboratory, we have screened microalgal strains producing high amount of carbohydrate compounds, and isolated a promising microalga strain NKY372003. In this study, we performed morphological observation and genotyping analysis, which indicates that this strain belongs to the algal genus Pseudoneochloris. Subsequently, we optimized the culture condition of this strain for high carbohydrate production by changing nutrient compositions of culture medium (e.g., nitrate and phosphate) and photon flux density. Based on the obtained results, we discussed
1389-1723/$ e see front matter Ó 2020, The Society for Biotechnology, Japan. All rights reserved. https://doi.org/10.1016/j.jbiosc.2019.12.010
Please cite this article as: Aketo, T et al., Characterization of a novel marine unicellular alga, Pseudoneochloris sp. strain NKY372003 as a high carbohydrate producer, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.12.010
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the potential of this strain as a host of carbohydrate production for biorefinery application.
MATERIALS AND METHODS Used strain Microalgal strains were isolated from seawater obtained from sites on the coast of Yakushima island, which is one of the Osumi islands in Kagoshima prefecture, Japan. Among 292 strains isolated, strain NKY372003 was selected as the highest carbohydrate producer for further study here. This strain was maintained in IMK medium (Nihon Pharmaceutical, Osaka, Japan) dissolved in artificial seawater in Erlenmeyer flasks (50 ml) at 100 mmol photons/m2/s and 30 C under the shaking condition. Microscopic analysis The morphologies of the cultured cells were analyzed by a differential interference contrast microscope (BX51; Olympus, Tokyo, Japan). Twenty cells were randomly selected and the diameters were measured by an image analysis software, ImageJ (13). To visualize intracellular starch granules, Lugol’s solution was prepared. Iodine crystals (50 mg) were dissolved in 1 ml of 100 mg/ ml potassium iodide solution. Subsequently, 10 ml of Lugol’s solution was added to 1 ml of cell suspension. Then, the cell suspension was heated at 98 C for 5 min to complete the staining (14). Phylogenetic analysis Strain NKY372003 was identified after analysis of the 18S rDNA sequence. Nucleic acid extraction was performed with DNeasy Tissue Kit (Qiagen, Venlo, Netherlands) in accordance with the manufacturer’s instructions. The 18S rDNA was amplified by PCR using a pair of universal primers, ss5 (50 -GGTGA TCCTG CCAGT AGTCA TATGC TTG-30 ) and ss3 (50 -GATCC TTCCG CAGGT TCACC TACGG AAACC-30 ) (15) and Takara LA Taq DNA polymerase (Takara Bio Inc., Shiga, Japan). The amplified fragments were inserted into pCR4-TOPO vector (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA). The resulting plasmids were sequenced using the following primers: Forward primers, T3 promoter-1 (50 ATTAA CCCTC ACTAA AGGGA-30 ), T7 promoter-1 (50 -TAATA CGACT CACTA TAGGG30 ) and 18SU467F (50 -ATCCA AGGAA GGCAG CAGGC-30 ); Reverse primers, 18SU1310R (50 -CTCCA CCAAC TAAGA ACGGC-30 ) (16). Sequence of 18S rDNA was determined by Fasmac DNA Sequence Service (Kanagawa, Japan). Sequence homologies were analyzed with BLAST. Evaluation of cell growth For the growth experiment, the full strength IMK nutrients, containing 2.4 mM nitrate and 0.038 mM phosphate, were dissolved in the artificial seawater (Osaka Yakken Co., Ltd., Osaka, Japan). We also prepared the IMK media with higher concentration of nitrate (4.7, 11.8 or 23.5 mM) by adding NaNO3, or higher concentration of phosphate (0.38 or 0.76 mM) by adding K2HPO4 and Na2HPO4 (weight ratio, 7:25, same with IMK medium). In addition, the condensed IMK media, 2IMK, 10IMK and 20IMK media, in which all nutrients were concentrated by 2-, 10-, and 20-fold as compare to the standard IMK medium, were prepared. Strain NKY372003 at the initial concentration of 1.0 105 cells/ml was cultured in each condition for 10 days in flat-shaped flasks (500 ml) at 30 C. Concentrated CO2 (2% in air) was supplied at the rate of 0.8 l/l/min for photoautotrophic culture. Furthermore, the effect of photon flux density on growth was evaluated. The cultured cells were collected at every 24 h, and growth was monitored by cell counting using hemocytometer. Three biological repetitions were analyzed for each sample. Measurement of biomass production, and carbohydrate and protein contents The cultured cells (5 ml) were collected at every 24 h. After centrifugation at 9000 g, 4 C for 10 min, the pellet was washed with 10 ml of deionized water. The algal biomass was lyophilized to measure the dry weight. Phenol-sulfuric acid method was employed to measure saccharide content (17). After the lyophilized cells were suspended in 500 ml of ultrapure water at the concentration of 0.5 mg/ml, 500 ml of 5% (w/v) phenol solution and 2.5 ml of concentrated sulfuric acid were added to the suspension. The samples were incubated for 30 min at room temperature. Subsequently, the absorbance at 490 nm was measured using a microplate reader (SH-9000; Corona Electric, Ibaraki, Japan). Protein quantity was analyzed by BCA Protein assay kit (Thermo Fisher Scientific). Absorbance at 562 nm was measured using the microplate reader. Measurement of nitrate concentration by HPLC Culture media were filtered using a membrane filter (Millex Filter Unit 0.22 mm). Nitrate concentrations in the filtered culture medium were determined using a Shimadzu Prominence HPLC system (Shimadzu Scientific Instruments, Kyoto, Japan) equipped with an anion exchange column, TSKgel IC column (Tosoh Co., Tokyo, Japan). TSKgel eluent ICAnion-A was used as a mobile phase at the flow rate of 1.2 ml/min to elute nitrate ion, and absorbance at 220 nm was measured by a UV detector (Shimadzu Scientific Instruments, Kyoto, Japan).
RESULTS AND DISCUSSION Characterization of strain NKY372003 by genotypic and morphological analyses As a result of the screening from 292 isolates, strain NKY372003 showed fast growth and the highest
carbohydrate content. Then, we examined the genotypic and morphological characteristics of this strain. Strain NKY372003 was identified based on 18S rDNA sequences (DDBJ/EMBL/GenBank accession number: LC505539), and displayed 96.95% of sequence identity with the 18S rDNA sequence of the unicellular green alga, Pseudoneochloris marina. Microscopic analysis indicated that vegetative cells of strain NKY372003 were spherical and nonmotile. Cell size was drastically changed during the growth phase, ranging from 3 mm to 15 mm in diameter (Figs. 1 and S1). Zoospores were produced in mature vegetative cells (Fig. S2A), and two flagella were located at the apex of the zoospore (Fig. S2B). Furthermore, Lugol staining indicates that starch granules were located and accumulated in the chloroplast (Fig. S3). These results were in agreement with the morphological data of P. marina (18). Therefore, this strain was tentatively identified as Pseudoneochloris sp. in this study.
Growth characteristics of Pseudoneochloris sp. NKY372003 at different nitrate and phosphate concentrations Fig. 2 shows the growth curves of Pseudoneochloris sp. NKY372003 in IMK medium (2.4 mM nitrate) at different photon flux densities. The final cell concentrations increased with increasing photon flux densities (Fig. 2A), and nitrates in the supernatants were completely consumed within 7 days in all the conditions (Fig. 2B). Although the final cell densities were different, the final biomass yield did not change markedly (Table S1). Especially, the biomass production at 800 mmol photons/m2/s was lower than those at 100 and 300 photons/m2/s, although the cell concentration at 800 mmol photons/m2/s was significantly higher than those at lower photon flux densities. This inconsistency could be due to smaller cell size at 800 mmol photons/m2/s than those at lower photon flux densities as mentioned above (Fig. S1). Decrease in cell size at 800 mmol photons/m2/s might be because of the phototoxicity at higher light intensities. These results indicated that light intensity was not a limiting factor for growth of strain NKY372003 in the standard IMK medium. Therefore, the effects of nutrients on algal growth was evaluated in the following experiments. Because the nitrogen and phosphorous (2.4 mM nitrate and 0.038 mM phosphate are contained in IMK medium) are essential elements for the growth of microalgae, effects of these nutrients on growth were firstly examined by adding them to standard IMK medium. It should be noted that pH values of the culture did not change largely. The initial pH of IMK medium was 7.27. After Pseudoneochloris sp. NKY372003 was cultivated in the IMK medium at 100 mmol photons/m2/s for 10 days, the pH value of the culture was 7.41 0.10. This could be due to the buffer action of the seawater-based culture medium. The growth of Pseudoneochloris sp. NKY372003 under the increased nitrate concentration (4.7 mM) at different light intensities (500 or 800 mmol photons/m2/s) was monitored. An increasing trend in cell concentrations was observed as increased nitrate concentration and light intensity (Fig. 3A). However, complete uptake of nitrate was not observed even at 4.7 mM, and the reductions of nitrate concentrations during the cultivation were comparable levels in all the tested conditions (Fig. 3B). As mentioned above, the cell concentrations were not exactly correlated to the biomass production in this experiment. Correspondingly, the biomass production did not change markedly by increasing the nitrate concentration and photon flux density (Table S2). Furthermore, the similar tendencies (i.e., increase in cell concentrations, but slight change in biomass production) were observed when increased phosphate concentrations were employed for the culture of Pseudoneochloris sp. NKY372003 (Table S3). Because sufficient CO2 (2%) was supplied in all the tested
Please cite this article as: Aketo, T et al., Characterization of a novel marine unicellular alga, Pseudoneochloris sp. strain NKY372003 as a high carbohydrate producer, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.12.010
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FIG. 2. Growth curve (A) and nitrate consumption (B) of Pseudoneochloris sp. NKY372003 cultured in standard IMK medium (2.4 mM nitrate and 0.038 mM phosphate) under different photon flux density (100e800 mmol photons/m2/s). Error bars indicate standard deviation (n ¼ 3).
FIG. 1. Microscope images of vegetative cells of Pseudoneochloris sp. NKY372003 cultivated in IMK medium for 10 days. Scale bars are 10 mm.
conditions, the medium components other than nitrogen and phosphorus were the limiting factor(s) of the biomass production. Evaluation of biomass production of Pseudoneochloris sp. NKY372003 in enriched culture media Sole increase in nitrate or phosphate in IMK medium did not significantly improve the biomass production of Pseudoneochloris sp. NKY372003. Then, in order to enhance the biomass production, enriched culture
media (i.e., concentrated 2 and 10IMK media) were tested under various light intensities. Only a representative experiment is shown in Fig. 4. Fig. 4A shows the growth curves of Pseudoneochloris sp. NKY372003 in 10IMK medium at 500 or 800 mmol photons/ m2/s of photon flux density. The cell concentrations in 10IMK medium was approximately twenty times higher than those in the standard IMK medium (Fig. 3A). It should be noted that the Yaxis in Fig. 4A was tenfold increase compared with that of Fig. 3A. Although much higher concentration of nitrate (23.5 mM) was contained in the condensed 10IMK medium, almost complete consumption of nitrate in the supernatants was observed in 5 days at both light conditions (Fig. 4B). As mentioned above,
Please cite this article as: Aketo, T et al., Characterization of a novel marine unicellular alga, Pseudoneochloris sp. strain NKY372003 as a high carbohydrate producer, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.12.010
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FIG. 3. Growth curve (A) and nitrate consumption (B) of Pseudoneochloris sp. NKY372003 cultured under different nitrate concentrations (2.4 or 4.7 mM) and photon flux density (500 or 800 mmol photons/m2/s). Error bars indicate standard deviation (n ¼ 3).
4.7 mM nitrate ions were not completely consumed in the standard IMK medium (Fig. 3B). By contrast, in 10IMK medium, additional nutrients could significantly accelerate the growth of Pseudoneochloris sp. NKY372003, and correspondingly enhance the uptake of nitrate ions in the enriched culture medium. The key components in the medium for this phenomenon should be elucidated in the future. The biomass production data using various media under different photon flux densities is summarized in Table 1. The use of 2IMK almost double in the biomass production compared with the standard IMK medium at 500 and 800 mmol photons/m2/s. Similarly, the use of 10IMK resulted in approximately 10- and 12-
J. BIOSCI. BIOENG.,
FIG. 4. Growth curve (A) and nitrate consumption (B) of Pseudoneochloris sp. NKY372003 in 10IMK medium at 500 or 800 mmol photons/m2/s. Error bars indicate standard deviation (n ¼ 3).
fold higher biomass production at 500 and 800 mmol photons/m2/s, respectively. These results indicate that the biomass production of Pseudoneochloris sp. NKY372003 was nutrient-dependent at these TABLE 1. Biomass production of Pseudoneochloris sp. NKY372003 cultured in the enriched IMK media at different photon flux density. Medium
Photon flux density (mmol photons/m2/s)
IMK 2IMK 10IMK IMK 2IMK 10IMK 10IMK
500 500 500 800 800 800 1300
Biomass production (g/L) 0.65 1.53 6.65 0.65 1.56 8.11 6.20
0.15 0.09 0.49 0.15 0.02 0.37
Please cite this article as: Aketo, T et al., Characterization of a novel marine unicellular alga, Pseudoneochloris sp. strain NKY372003 as a high carbohydrate producer, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.12.010
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obviously repressed the cell growth in 10IMK, resulting in lower biomass production (6.20 g/L). This might be also caused by the phototoxicity at higher light intensities. In addition, 20IMK was used for the growth test, however, no cell growth was observed (data not shown). In microalgae, cell growth can be inhibited due to excessive nutrients such as nitrate, nitrite, and ammonium (19,20). In C. vulgaris, growth inhibition was observed at more than 29 mM nitrate (20). The increase of intracellular nitrate may induce high accumulation of nitrite and/or ammonium by nitrate reductase, resulting in the growth inhibition by the accumulated toxic compounds (20). The similar growth inhibition could occur in this experiment because the condensed 20IMK medium contained much higher nitrate concentration (47 mM nitrate) than that previously reported. Based on the above results, carbohydrate production in strain NKY372003 was further investigated in 10IMK medium in the following experiments.
FIG. 5. Time course variations of carbohydrate (closed bars) and protein (open bars) contents, and biomass production (closed circles) of Pseudoneochloris sp. NKY372003 in 10IMK medium at (A) 500 or (B) 800 mmol photons/m2/s. Error bars indicate standard deviation (n ¼ 3).
conditions, although the nutrients which indeed limited the growth were not specified in this study. The maximum biomass production was 8.11 0.37 g/L at 800 mmol photons/m2/s when 10IMK was used. Higher light (1300 mmol photons/m2/s)
Evaluation of carbohydrate production of Pseudoneochloris sp. NKY372003 Fig. 5 shows time-courses variations of carbohydrate and protein contents, and biomass productions in 10IMK medium at 500 or 800 mmol photons/m2/s. Remarkable carbohydrate accumulation was observed after day 6. Because nitrate consumption in 10IMK medium was almost completely consumed at day 5 (Fig. 4B), the carbohydrate accumulation could be induced by nitrogen-deficient stress. It was frequently reported that nitrogen starvation induced carbohydrate accumulation in microalgal cells (21,22). In contrast, the protein contents tended to decrease with time. Nitrogen is an important component for the synthesis of amino acids (proteins), therefore, the tradeeoff relationship between carbohydrate and protein contents could be a response to the nitrogen deficiency, generally observed in microalgae (23,24). It should be noted that obvious decrease in biomass production was repeatedly observed at day 7 at 800 mmol photons/m2/s (Fig. 5B). The phenomenon will be elucidated in detail in the future, but the zoospore formation (Fig. S2A) from vegetative cells will be a possible reason for the weight loss. Both carbohydrate content and biomass production were maximum at 800 mmol photons/m2/s (67.8 7.0%, and 8.11 0.37 g/L, respectively) at day 10. The resulting carbohydrate production reached to 5.5 0.2 g/L. Recently, biochemical compositions of P. marina, the closely related microalga, cultivated under various conditions was reported (21). According to this literature, maximal biomass production and carbohydrate content of P. marina reached 2.02 0.21 g/L and 53.8% in 7 days, respectively, suggesting that carbohydrate production in P. marina was less than 1.09 g/L. Carbohydrate production of representative microalgae is summarized in Table 2. As far as we know, carbohydrate production by Pseudoneochloris sp. NKY372003 was the highest level among ever reported. The carbohydrate productivity per unit time of Pseudoneochloris sp. NKY372003 was 0.55 0.08 g/L/day, which were higher than that of P. marina (<0.16 g/L/day) (21), and comparable to that of Tetraselmis subcordiformis (0.20e0.62 g/L/day) (22).
TABLE 2. Comparison of biomass and carbohydrate production among various microalgal species. Biomass production (g/L) Chlorella vulgaris Chlamydomonas reinhardtii Scenedesmus obliquus Arthrospira platensis (Spirulina platensis) Synechococcus sp. PCC7002 Tetraselmis subcordiformis Pseudoneochloris marina Pseudoneochloris sp. NKY372003
Carbohydrate production (g/L)
Reference
0.5e7.3 1.5e2.4 2.6e4.0 2.0e2.2
0.3e3.6 0.8e1.2 1.3e1.9 1.2e1.3
23,25,26 27,28 23,29 30
3.0 4.5e6.0 1.33e2.02 8.11 0.37
1.8 1.8e3.0 <1.09 5.5 0.2
31 22,32 21 This study
Please cite this article as: Aketo, T et al., Characterization of a novel marine unicellular alga, Pseudoneochloris sp. strain NKY372003 as a high carbohydrate producer, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.12.010
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As mentioned above, Pseudoneochloris sp. NKY37200 accumulated starch granules intracellularly. Starch can be easily converted to fermentable monosaccharides like glucose, and subsequently such monosaccharides can be transformed into bioethanol and biobutanol by a fermentation process. Furthermore, recently, algal starch can be successfully fermented to building blocks, such as succinate, methyl levulinate or methyl lactate, useful for the synthesis of fine chemicals (11,12). Therefore, Pseudoneochloris sp. NKY372003 will be a promising candidate for production of fermentable sugar towards biofuels and fine chemicals production. In conclusion, microalga Pseudoneochloris sp. NKY372003 was newly identified as a high carbohydrate producer. Elevation of entire concentration of IMK medium drastically increased the biomass production, which reached the maximal value of 8.11 0.37 g/L when 10IMK medium were used for cultivation at 800 mmol photons/m2/s. In this condition, carbohydrate content and production were 67.8 7.0% and 5.5 0.2 g/L, respectively. To the best of our knowledge, this is the highest carbohydrate production by microalgae. These results indicate that Pseudoneochloris sp. NKY372003 is a promising host of carbohydrate production for biorefinery applications. Supplementary data to this article can be found online at https://doi.org/10.1016/j.jbiosc.2019.12.010. ACKNOWLEDGMENTS This work was partially supported by JSPS KAKENHI Grant-inAid for Scientific Research B [grant number 17H03465]. References 1. Lahaye, M. and Robic, A.: Structure and functional properties of ulvan, a polysaccharide from green seaweeds, Biomacromolecules, 8, 1765e1774 (2007). 2. Bikker, P., van Krimpen, M. M., van Wikselaar, P., Houweling-Tan, B., Scaccia, N., van Hal, J. W., Huijgen, W. J., Cone, J. W., and LopezContreras, A. M.: Biorefinery of the green seaweed Ulva lactuca to produce animal feed, chemicals and biofuels, J. Appl. Phycol., 28, 3511e3525 (2016). 3. Glasson, C. R., Sims, I. M., Carnachan, S. M., de Nys, R., and Magnusson, M.: A cascading biorefinery process targeting sulfated polysaccharides (ulvan) from Ulva ohnoi, Algal Res., 27, 383e391 (2017). 4. Chisti, Y.: Biodiesel from microalgae, Biotechnol. Adv., 25, 294e306 (2007). 5. Maeda, Y., Yoshino, T., Matsunaga, T., Matsumoto, M., and Tanaka, T.: Marine microalgae for production of biofuels and chemicals, Curr. Opin. Biotechnol., 50, 111e120 (2018). 6. Chen, C. Y., Zhao, X. Q., Yen, H. W., Ho, S. H., Cheng, C. L., Lee, D. J., Bai, F. W., and Chang, J. S.: Microalgae-based carbohydrates for biofuel production, Biochem. Eng. J., 78, 1e10 (2013). 7. Kim, K. H., Choi, I. S., Kim, H. M., Wi, S. G., and Bae, H. J.: Bioethanol production from the nutrient stress-induced microalga Chlorella vulgaris by enzymatic hydrolysis and immobilized yeast fermentation, Bioresour. Technol., 153, 47e54 (2014). 8. Moncada, J., Jaramillo, J. J., Higuita, J. C., Younes, C., and Cardona, C. A.: Production of bioethanol using Chlorella vulgaris cake: a technoeconomic and environmental assessment in the colombian context, Ind. Eng. Chem. Res., 52, 16786e16794 (2013). 9. Choi, S. P., Nguyen, M. T., and Sim, S. J.: Enzymatic pretreatment of Chlamydomonas reinhardtii biomass for ethanol production, Bioresour. Technol., 101, 5330e5336 (2010). 10. Osanai, T., Shirai, T., Iijima, H., Nakaya, Y., Okamoto, M., Kondo, A., and Hirai, M. Y.: Genetic manipulation of a metabolic enzyme and a transcriptional regulator increasing succinate excretion from unicellular cyanobacterium, Front. Microbiol., 6, 1064 (2015). 11. Yamaguchi, S., Kawada, Y., Yuge, H., Tanaka, K., and Imamura, S.: Development of new carbon resources: production of important chemicals from algal residue, Sci. Rep., 7, 855 (2017).
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Please cite this article as: Aketo, T et al., Characterization of a novel marine unicellular alga, Pseudoneochloris sp. strain NKY372003 as a high carbohydrate producer, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.12.010